Kinetics of antigen-specific CD4⁺ T cell expansion after <i>C. muridarum</i> intravaginal (i.vag.) infection.

<p>(A) C57BL/6 mice were immunized subcutaneously with 100 µg of <i>C. muridarum</i> peptide (RplF_51–59, Aasf_24–32 or PmpG-1_303–311) in the presence of CFA or infected intravenously with either 1×10⁵<i>C. muridarum</i> or 5×10⁵<i>S. typhimurium</i>. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from <i>C. muridarum</i> or <i>S. Typhimurium</i> infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF_51–59:I-A^b, Aasf_24–32:I-A^b or PmpG-1_303–311:I-A^b tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b⁻CD11c⁻F4/80⁻B220⁻CD3⁺CD4⁺ cells except CD8⁺ T cells (bottom row), which were gated on CD11b⁻CD11c⁻F4/80⁻B220⁻CD3⁺CD8⁺ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×10⁵<i>C. muridarum</i>. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1_303–311:I-A^b tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b⁻CD11c⁻F4/80⁻B220⁻CD3⁺CD4⁺ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.